의 동기화<em> Caulobacter Crescentus</em> 세균 세포주기의 조사에 대한
Summary
Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.
Abstract
The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.
Introduction
박테리아 세포 사이클은 게놈의 복제와 딸 세포 분열의 양을 제어한다. 항생제 내성은 공중 보건에 대한 위협도 갈수록 증가로 중요한 것은, 세균 세포주기 항생제 개발을위한 미개척 목표를 제시한다.
세균 Caulobacter crescentus의, 각 셀은 서로 다른주기 운명 (도 1a)의 1,2- 개의 딸 세포를 수득 비대칭 분할 리드. 한 딸 셀은 편모를 상속하고 다른 딸이 줄기를 상속 고착 동안 운동성이있다. 통합 유전자 회로는 전사 조절, 인산화 신호 전달 및 조절 단백질 분해 (3)에 의한 세포주기 진행과 세포의 운명을 제어합니다. 또한, 염색체 복제 및 동시 분리 수율 딸 세포는 염색체 4의 단 하나의 복사본을 포함하고있다. 중요한 것은, 이러한 두 가지 종류의 세포는 급속 collo 의해 분리 될 수있다높은 수율 (도 1b)와 인구의 나머지 swarmer 세포의 분리를 허용하는 동기화 가능한 균주의 NA1000 5-7 idal 실리카 입자 밀도 원심. 세포 swarmer 절연 한 후 비대칭 세포 분열을 통해 동 기적으로 진행합니다. 여기서 우리는 세부 프로토콜은 Caulobacter 변형 NA1000를 동기화에 사용됩니다. 우리는 프로토콜과의 크고 작은 규모 모두 동기화에 대한 일반적인 문제 해결 팁을 제공합니다. 이 실험 절차는 Caulobacter의 세포주기 및 세포 운명의 시공간 제어를 심문 할 수있는 강력한 도구를 제공합니다.
Protocol
Representative Results
Discussion
The bacterial cell cycle is a fundamental process in life and is important for the study of growth and as a target for next generation antibiotics. Here, we detailed the rapid synchronization procedures for C. crescentus NA1000, a model organism for the study of the bacterial cell cycle and asymmetric cell division. This method is amendable to western blot, gene expression profiling, and fluorescence microscopy assays to investigate the spatiotemporal regulation of the bacterial cell cycle.
<p class='jove_…Divulgations
The authors have nothing to disclose.
Acknowledgements
The authors thank members of the Shapiro lab and Erin Schrader for comments on the manuscript. The authors acknowledge financial support from: NIH postdoctoral fellowship F32 GM100732 to JMS and NIH grants R01 GM51426 and R01 GM32506 to LS.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| PVP Coated Colloidal Silica (Percoll) | Sigma-Aldrich | P4937 | |
| Colloidal Silica (Ludox AS-40) | Sigma-Aldrich | 420840 | |
| JA10 Rotor | Beckman-Coulter | 369687 | |
| JA20 Rotor | Beckman-Coulter | 334831 | |
| Ferrous Sulfate Chelate Solution | Sigma-Aldrich | F0518 | |
| 30 mL Centrifuge Tubes | Corning | 8445 | |
| Na2HPO4 | EMD | SX0720-1 | |
| KH2PO4 | VWR | BDH9268-500G | |
| NH4Cl | Amresco | 0621-500g |
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